ovalbumin and pyrene

Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml(-1). The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples.

Topics: Animals; Antibodies, Monoclonal; Calibration; Cattle; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Limit of Detection; Linear Models; Ovalbumin; Pyrenes; Serum Albumin, Bovine; Water

Novel strategies of developing fluorescent sensors for proteins are highly demanded. In this work, we particularly synthesized a cholesterol-derivatized pyrene probe. Its fluorescence emission is effectively tuned by the aggregation state of a cationic surfactant dodecyltrimethylammonium bromide (DTAB). The used probe/DTAB assemblies exhibit highly sensitive ratiometric responses to pepsin and ovalbumin egg (o-egg) with detection limits of 4.8 and 18.9 nM, respectively. The fluorescence changes indicate the protein-surfactant interaction leads to further aggregation of DTAB assemblies. The results from Tyndall effect and dynamic light scattering verify this assumption. The responses to pepsin and o-egg are due to their strong electrostatic or hydrophobic interaction with DTAB assemblies at pH 7.4. The present noncovalent supramolecular sensor represents a novel and simple strategy for sensing proteins, which is based on the encapsulated fluorophore probing the aggregation variation of the surfactant assemblies.

Topics: Cholesterol; Fluorescent Dyes; Micelles; Ovalbumin; Pepsin A; Protein Binding; Pyrenes; Quaternary Ammonium Compounds; Spectrometry, Fluorescence; Surface-Active Agents; Water

pH-Sensitive dextran derivatives having 3-methylglutarylated residues (MGlu-Dex) were prepared by reacting dextran with 3-methyl-glutaric anhydride. MGlu-Dex changed the protonation state and their characteristics from hydrophilic to hydrophobic in neutral and acidic pH regions. Surface modification of egg yolk phosphatidylcholine liposomes with MGlu-Dex produced highly pH-sensitive liposomes that were stable at neutral pH but which were destabilized strongly in the weakly acidic pH region. MGlu-Dex-modified liposomes were taken up efficiently by dendritic cells and delivered entrapped ovalbumin (OVA) molecules into the cytosol. When MGlu-Dex-modified liposomes loaded with OVA were administered subcutaneously to mice, the antigen-specific humoral and cellular immunity was induced more effectively than the unmodified liposomes loaded with OVA. Furthermore, administration of MGlu-Dex-modified liposomes loaded with OVA to mice bearing E.G7-OVA tumor significantly suppressed tumor growth and extended the mice survival. Results suggest that MGlu-Dex-modified liposomes are promising for the production of safe and potent antigen delivery systems that contribute to the establishment of efficient cancer immunotherapy.

Topics: Anhydrides; Animals; Antigens, Neoplasm; Cell Line; Chemical Precipitation; Dextrans; Female; Fluorescence; Fluorescent Dyes; Glutarates; Hydrogen-Ion Concentration; Immunity; Immunization; Immunotherapy; Interferon-gamma; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Particle Size; Pyrenes; Static Electricity; Subcutaneous Tissue; T-Lymphocytes, Cytotoxic; Titrimetry

Here we present an efficient synthesis of functional dendritic polymers carrying internal fluorescence labels for bioconjugation. Specifically, dendritic polymers having pyrene as fluorescence label in the core and N-hydroxysuccinimide (NHS) functional groups at the periphery were synthesized by coupling heterobifunctional PEG to hydroxyl functionalized dendritic polyethylene core. The dendritic polyethylene cores containing one pyrene label per polymer molecule were prepared through a one-step transition-metal-catalyzed polymerization using a pyrene-labeled Pd(II)-alpha-diimine chain walking catalyst. A series of pyrene-labeled dendritic scaffolds were obtained with different molecular weights and sizes. NHS active end groups were introduced to the periphery of the dendritic scaffolds through end-group functionalization. Those NHS-functionalized dendritic scaffolds were successfully used to conjugate a model protein, ovalbumin, to yield protein-polymer conjugates carrying multiple copies of protein attached to each scaffold.

Topics: Catalysis; Cross-Linking Reagents; Dendrimers; Fluorescence; Molecular Probe Techniques; Ovalbumin; Proteins; Pyrenes

Since antigen-specific autoaggressive T cells have been found in association with many autoimmune diseases, a treatment to eliminate such antigen-specific T cell clones was developed. The complex of peptide antigen and class II MHC protein is used to target a cytotoxic drug to antigen-specific T cells. The drug is bound covalently to antigen-presenting cells (APC) and protein antigens (Ag) are added to the cells for processing and presentation of peptides. The APC contain class II MHC (Ia) protein to present the peptide Ag to the T cell receptor and adhesion proteins for optimal interaction with the T cell. Either the Ag-bearing intact APC or Ia+ membranes shed or released spontaneously from them were used as drug carriers to target the drug to the T cells. The drugs being used are phototoxic compounds. When irradiated with light of an appropriate wavelength, they give off toxic free radicals and singlet oxygen. These toxic by-products are short-lived and damage cells only in their immediate vicinity, cutting down on nonspecific side effects. APC from thymus cells, or their shed membranes bearing Ia-Ag peptide complexes, were able to target the phototoxic drug specifically to Ag-specific T line cells and ex vivo Ag-specific lymph node cells. Proliferation of the target T cells was inhibited at a three to four times lower drug concentration than required to affect control T cells. The Ag-specific effect was inhibited by anti-Ia antibody and by drug-free membranes carrying the Ag-Ia complex. This indicated that the antigen-specific phototoxic effect was mediated by interaction of the Ag-Ia complex with the T cell receptor.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antigen Presentation; Antigen-Presenting Cells; Binding, Competitive; Cell Line; Cell Membrane; Clonal Deletion; Epitopes; Hemocyanins; Histocompatibility Antigens Class II; Immunization; Lymph Nodes; Myelin Basic Protein; Ovalbumin; Pyrenes; Rats; Rats, Inbred Lew; Receptors, Antigen, T-Cell; Staining and Labeling; T-Lymphocytes

We have previously demonstrated that pyrene in diesel-exhaust particles (DEP) has an adjuvant activity on immunoglobulin E (IgE) antibody production in mice immunized with Japanese cedar pollen allergen (JCPA) or ovalbumin (OA) intraperitoneally. The present study is concerned with the adjuvant activity in IgE antibody production against JCPA of pyrene or DEP inoculated intranasally in mice. We show that anthracene, fluoranthene and benzo(a)pyrene in DEP have the ability to enhance anti-JCPA IgE antibody production in mice by intranasal immunization. Mice were grouped, immunized with 10 micrograms of JCPA plus 400 micrograms of pyrene, 10 micrograms of JCPA plus 100 micrograms of DEP, 10 micrograms of JCPA plus 2 mg of aluminum hydroxide and 10 micrograms of JCPA alone intranasally 7 times at 2 week intervals. Mice were also grouped, and immunized with JCPA (10 micrograms) plus 40 micrograms of anthracene, JCPA (10 micrograms) plus 400 micrograms of fluoranthene, JCPA (10 micrograms) plus 40 micrograms of benzo(a)pyrene, and JCPA (10 micrograms) plus 400 micrograms of pyrene and JCPA (10 micrograms) alone. We found that the IgE antibody responses to JCPA in mice immunized with JCPA plus pyrene, JCPA plus DEP or JCPA plus the three chemical organic compounds mentioned above were significantly enhanced compared with those immunized with JCPA alone. In addition, when the intraperitoneal macrophages obtained from the normal mice (unimmunized mice) were incubated with pyrene, anthracene, fluoranthene or benzo(a)pyrene in vitro, an enhanced chemiluminescence (CI) response and interleukin-1 alpha (IL-1 alpha) production of the macrophages was observed in each instance. These results suggest that in the production of IgE antibody to JCPA the adjuvancy of polycyclic aromatic hydrocarbons (PAHs) in DEP may be important in an attack of Japanese cedar pollinosis.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Air Pollutants; Allergens; Aluminum Hydroxide; Animals; Anthracenes; Antibodies, Anti-Idiotypic; Benzo(a)pyrene; Female; Fluorenes; Food Contamination; Humans; Immunization; Immunoglobulin E; Interleukin-1; Luminescent Measurements; Macrophages, Peritoneal; Male; Mice; Ovalbumin; Pollen; Polycyclic Aromatic Hydrocarbons; Pyrenes; Rats; Rats, Wistar; Rhinitis, Allergic, Seasonal; Trees; Urban Population; Vehicle Emissions

In this communication, it is shown that pyrene has an adjuvant activity on IgE antibody production when mice are immunized by an intraperitoneal injection of ovalbumin (OA) or Japanese cedar pollen allergen (JCPA) with pyrene. The effects of pyrene on IgE antibody production in mice were investigated to clarify the relation between pollen allergy and the adjuvanticity of the chemical compounds contained in diesel-exhaust particulates (DEP). In the first experiment, three groups of mice were immunized intraperitoneally six times at 2-week intervals with 1 microgram of OA alone, 1 microgram of OA plus 1 mg of pyrene, and 1 microgram of OA plus 1 mg of DEP, respectively. The IgE antibody responses to OA in mice immunized with OA plus pyrene or OA plus DEP were extremely enhanced as compared with those in mice immunized with OA alone, and the highest responses were observed in mice immunized with OA plus DEP. In the second experiment, mice were immunized with 10 micrograms of JCPA alone or 10 micrograms of JCPA plus 5 mg of pyrene in the same way. The IgE antibody responses to JCPA in mice immunized with JCPA plus pyrene were higher than those in mice immunized with JCPA alone. The intraperitoneal macrophages of the mice also clearly stimulated in vitro by pyrene on chemiluminescence assay. These results suggest that pyrene contained in DEP acts as an adjuvant in IgE antibody production when mice are immunized with antigens.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Cells, Cultured; Female; Immunoglobulin E; Male; Mice; Ovalbumin; Pollen; Pyrenes; Vehicle Emissions